Enzyme immobilization in a gel containing 30 to 50 percent gelatin

ABSTRACT

Gel immobilized enzymes are prepared for use in carrying out reactions in hydrophobic solvents. The gel is formed from a gelatinizer such as gelatin or a polysaccharide such as agarose, agar, pectin, sodium alginate or carrageenan. The gel contains a ratio of amount of gelatinizer to amount of water such that the gel is capable of being mechanically divisible into dimensionally substantially stable fragments at a temperature which may reach a lower limit of the gelation temperature range of the gelatinizer. A preferred gel contains 30 to 50% gelatin, and is prepared by forming a mixture of water, enzyme and water-soluble gelatin, heating the mixture to dissolve the gelatin to form a solution, and cooling the solution until it forms a gel. An immobilized lipase can be used for synthesis of an optically active isomer of an ester from an alcohol and an acid, with either the alcohol or the acid being a racemic mixture or an enantiomer, for hydrolysis of an ester which is either a racemate or a pure enantiomer, or for transesterfication.

This application is a U.S. national stage of International ApplicationPCT/FI97/00489, filed Aug. 25, 1997, and published on Mar. 5, 1998 inthe English language.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a gel containing an immobilised enzyme,a method for preparing such a gel and its application inenzyme-catalysed reactions in hydrophobic solvents.

2. Description of the Related Art

Enzyme-catalysed reactions are in widespread use particularly infoodstuffs and pharmacological chemistry. Some enzymes being veryexpensive, it is essential that they are carefully recovered for re-usein subsequent reactions. This can be done by immobilising the enzymes onsolid supports e.g. in the form of small solid beads which areintroduced in the reaction mixture and separated after the reaction hasbeen accomplished. Continuous reactions may be performed by placingthese beads in a column and by allowing the reaction mixture to flowthrough the bed of beads. In such beads, the enzyme is covalently boundinto a compound, e.g. a polymer containing reactive groups reacting withthe amino groups in the protein part of the enzyme.

The work Koskinen A. M. P. & Klibanov, A. M. Blackie Academic &Professional, Glasgow 1996 describes methods for immobilising an enzymeon a solid support in the section “Modes of using enzymes in organicmedia” by P. Adlercreutz. The methods may be summarised as follows:

an aqueous solution containing the enzyme is brought into contact with asolid support, water is removed under reduced pressure and the enzyme isprecipitated onto the solid support;

the enzyme is precipitated from an aqueous solution in the presence of asolid support, the precipitation taking place with the addition of acooled, water-miscible solvent, such as acetone;

the enzyme in the aqueous solution is allowed to spontaneously adsorb ona solid support;

the enzyme is covalently bound to a solid support, and

the enzyme, which is adsorbed or precipitated on a solid support, iscross-linked with glutaraldehyde.

The publication S. Backlund et al., Kamia-Kemi Vol. 20 (1993) 197-201describes an enzyme-catalysed synthesis of a number of esters by meansof the enzyme lipase incorporated in a water-in-oil microemulsion. Thepublication S. Backlund et al., Colloid Polym Sci 274:540-547 (1996)describes a lipase-catalysed synthesis of optically active esters fromracemic 2-octanol and various carboxylic acids. The lipase isincorporated in a microemulsion-based gel, in which the microemulsion iseither a water-in-oil microemulsion or a microemulsion having abicontinuous structure.

However, the above methods for immobilising enzymes involve a largenumber of drawbacks. Above all, the immobilising methods described aboveare very sophisticated, laborious and expensive.

Precious enzymes are used e.g. in the preparation of optically activepharmaceutical compounds, which obviously have to be perfectly pure andabove all, free from toxic substances. In this conjunction, polymers andsimilar enzyme-binding substances may involve an undesired risk.Hydrocarbons are for instance used as a hydrophobic component in thepreparation of microemulsions. Surfactants are used to stabilisemicroemulsions, AOT (sodium 1,4-bis (2-ethylhexyl)sulfosuccinate) beingprobably the most common of these. The AOT surfactant is not suitablefor use in pharmaceutical contexts.

EP patent application 68594 describes a gel consisting of water, enzyme,gelatiniser, buffer, and albumine or protein. The albumine or proteincomponent is allegedly added to stabilise the enzymic system. The gelthus produced will have the form of small beads. Judging by thedisclosure, the mixture used is unlikely to have resulted in a gel whichis mechanically divisible into fragments at a temperature equalling atleast approximately the gelatination temperature of the gelatiniser orat the ambient temperature. The disclosure indicates that the mixturewas cooled down to 5° C. Cooling to such a low temperature implies thatbead formation would have been impossible at a higher temperature.

The publication Biotech. Bioeng. 21 (1979) 1697-1709 (Tosa et al)describes a gel with carrageenan as a gelatiniser. The gel was shaped asa cube at a low temperature (10° C.). There is no suggestion of thepossibility to divide this gel mechanically at the gelatinationtemperature of carrageenan or in the proximity of this.

SUMMARY OF THE INVENTION

The purpose of the present invention is to achieve a straightforward gelproviding ease of production and handling and containing an enzymeimmobilised within it. A further purpose is that such a gel be free fromtoxic substances and thus suitable for pharmaceutical synthesis.Moreover, none of the components in the gel should act as a substratefor the enzyme. Still a further purpose is to provide a gel having asurface with open pores such that the enzyme immobilised within the gelis well contacted with the surrounding reaction mixture. The purpose isalso to enable the reaction to be performed at the ambient temperaturewithout stirring, which is an economical procedure in terms of energysupply.

Thus, the invention relates to a gel containing an immobilised enzyme.The gel is characterised in that it consists of a water-soluble enzyme,a water-soluble gelatiniser and water, the ratio of the amount ofgelatiniser to the amount of water having been selected such that thegel formed can be mechanically divided into dimensionally substantiallystable fragments at a temperature which can reach the lower limit of thegelatination temperature range of the gelatiniser.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of optically active isomers of2-octylhexanoate from racemic 2-octanol and hexanoic acid as a functionof time, with the use of four mutually identically gels (A-D) containinggelatin, lipase and water.

FIG. 2 shows the synthesis as a function of time with the use of asingle gel in four successive syntheses I to IV.

FIG. 3 shows the synthesis as a function of time with the use of threegels (PAG1, PAG5 and PAG10) containing different amounts of enzyme.

FIG. 4 shows the synthesis as a function of time with the use of threegels having different gelatin concentrations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following, the term “immobilising” implies that the enzyme isimmobilised by being dissolved in a hydrogel, i.e. a gel with water asthe solvent.

The wording “mechanically divisible into dimensionally substantiallystable fragments” signifies that one should be able to cut the gel intofragments which can be handled and conserved without losing their easeof handling during storage. Likewise, it is crucial that this gel willremain intact in the hydrophic solvent in which the gel is to be used.

Substances suitable as gelatinisers are for instance gelatin orpolysaccharides such as agarose, agar, pectin, sodium alginate orcarrageenan.

With gelatin as a gelatiniser, the gel will have a 20 to 50% gelconcentration, preferably 30 to 44%. The corresponding ranges for agarand sodium alginate are 5 to 20% and 10 to 15%, respectively.

The gel of this invention is prepared by adding to the water-solublegelatiniser an amount of water selected such that the gel formed will bemechanically divisible into dimensionally substantially stable fragmentsat a temperature that may reach the lower limit of the gelatinationtemperature range of the gelatiniser. The mixture obtained is heated toa temperature at which the gelatiniser dissolves in water (approx. 50°C. for gelatin and approx. 90° C. for agar), upon which the solutionthus obtained is cooled until it is gelatinised.

Most gelatinisers have a gelatination temperature in the range of 30 to40° C. Sodium alginate is gelatinised by adding Ca²⁺ ions and this isthe reason why they are appropriately prepared at the ambienttemperature e.g. in a 0.05 M CaCl₂ solution.

Even though the gels of the invention can be cut into dimensionallystable fragments at temperatures which may reach the lower limit of thegelatination temperature range of the gelatiniser, in practicalimplementation, the gel is appropriately cut at the ambient temperatureor at a somewhat lower temperature (approx. 18 to 25° C.).

The gel is used in enzyme-catalysed reactions in which the reactants aredissolved in a hydrophobic solvent. Gel fragments containing the gel arecontacted with the reactants, the reaction occurring at the interfacebetween the gel and the hydrophobic solvent. Porous gel fragments willbe particularly suitable, since the reaction solution penetrates intothe pores of the gel fragments. This increases the interface at whichthe enzyme-catalysed reaction takes place.

With lipase as the immobilised enzyme, the gel will be suitable forsynthesis of optically active esters from racemic alcohols andcarboxylic acids, or for the preparation of optically active alcohols orcarboxylic acids by hydrolysis of racemic esters, or forreesterifications.

The invention will be described in greater detail below by means of anembodiment example. All of the experiments comprised synthesis ofoptically active isomers of 2-octylhexanoate from racemic 2-octanol andhexanoic acid. The test results are illustrated with figures

EXAMPLE

Materials

The following reactants were used in the experiments: gelatin type B,710 μm, approx. Bloom 230 supplied by the University Pharmacy of Turku,(±)-2-octanol (approx. 98%) and hexanoic acid (>98%) from Fluka, hexane(p.a.) from Merck, 4-dimethylaminopyridine (DMAP), acetic acid anhydride(>98%) and pyridine (HPLC degree) from Sigma Chemicals, and lipaseCandida sp. (SP525) from Novo Nordisk, Denmark. (±)-2-oxtylhexanoate(approx. 95%) was chemically synthesised. The water was distilleddeionised.

Methods

a) Preparation of gel

The gelatin and the water into which the lipase had just been dissolvedwere heated to 50° C. while stirring for about 5 to 7 minutes to makethe gelatin dissolve. The solution was stabilised at 30° C. for onehour, after which the pseudosolid aquatic gel (PAG) was conserved at−20° C. until it was used.

b) Reaction

The pseudosolid gel (PAG) was cut into about 25 fragments at the ambienttemperature and was transferred into a 100 ml E retort. A hexanesolution containing 0.33 M (±)-2-octanol and 0.33 M hexanoic acid forsynthesis (or 0.33 M (±)-2-oxtyl hexanoate for hydrolysis) wasintroduced in the retort, the reaction being then initiated. Thereactions were performed at 25° C. without shaking the reaction retort.

c) Sampling and analysis

At the end of given intervals samples (usually of 0.150 cm³) were takenfrom the reaction solution in order to monitor the reaction process. Thesamples were acetylised with acetic acid anhydride. DMAP dissolved inpyridine acted as a catalyst. The acetylised samples were analysed bymeans of a Varian 3400 gas chromatograph, to which a chiral column(Cyclodex-B from J & W Scientific; 30 m×0.252 mm with a film thicknessof 0.25 μm) was coupled.

Results

Conversion

The areas for the reactants or substrates (s) and the reaction products(p) were read from the chromatograms. The conversion (c) was calculatedwith the formula

c=ee_(s)/(ee_(c)+ee_(p))

where ee_(c)=(S1−S2)/(S1+S2) and ee_(p)=(P1−P2)/(P1+P2). S1 or P1corresponds to the area of the dominating enantiomer, while S2 or P2corresponds to the area of the second enantiomer.

Reproducibility

Comparison of the synthesis process of four separate pseudosolid gels(A, B, C, D) with identical composition: The gelatin amount was 1.4 gand the water amount 1.8 g. All of the reactions reach a 0.45 conversionat the end of about 12 days, when equilibrium is achieved. A and Bproduce almost identical processes, whereas C and D have a somewhatlower initial rate. The results are presented in Table 1 and FIG. 1.

TABLE 1 Reproducibility. Gels A-D contained 43.8% of gelatin and 8.8 mgof lipase. Pseudosolid gel Time (h) when c = 0.2 A 48 B 48 C 59 D 75

Re-use of the gels

The same pseudosolid gel was used four subsequent times in identicalsyntheses (I, II, III, IV). The gel was repeatedly rinsed with hexanebetween the cycles. The same conversion degree is probably obtained,however, the reaction will slow down for each re-use. The gel will alsohave caked to some extent. The results are shown in Table 2 and FIG. 2.

TABLE 2 Re-use. Gel composition: 43.8% of gelatin and 8.8 mg of lipase(1.4 g of gelatin and 1.8 g of water). Synthesis number Time (h) when c= 0.2 I 48 II 54 III 72 IV 87

Enzyme amount

Pseudosolid gels with identical compositions (gelatin:water) but withdifferent enzyme concentrations were mutually compared in a synthesis ofthe type described above. PAG1 contained 8.8 mg, PAG5 44 mg and PAG10 88mg of lipase. The greater the amount of added enzyme, the faster the0.45 conversion degree was achieved. The results are shown in Table 3and FIG. 3.

TABLE 3 Impact of the enzyme amount. The gels contained 43.8% of gelatin(1.4 g of gelatin and 1.8 g of water). Enzyme amount Time (h) Time PAGmg when c = 0.2 Max c days ea_(s) ee_(p) 1 8.8 48 0.45 12  0.80 0.99 544 19 0.45 8 0.80 0.99 10  88 12 0.45 6 0.80 0.99

Gelatin amount

Pseudosolid gels having different gelatin concentrations (43.8%, 30%,20%) were tested in syntheses of the kind described above. Thepseudosolid gel becomes softer, the lower the gelatin concentration is.In hexane without substrates, gels with a less than 20% gelatinconcentration remain stable, whereas used with a substrate, the gelpieces will cake relatively fast even for 20 and 30% gelatin, forming amass full of lumps. The reaction is faster in softer pseudosolid gels,which, however, are also much faster destroyed. The results are shown inTable 4 and FIG. 4.

TABLE 4 Impact of the gelatin concentration. The gels contained 8.8 mgof lipase (gelatin 1.4 g). Gelatin Time Time Obser- PAG % when c = 0.2Max c days vations a 43.8 48 0.45 12 pieces b 30 32 0.45 ≧15 lumps c 2021 0.45 >15 thick mass

Reversibility

The hydrolysis conversion degree was examined by using the ester as asubstrate. At the end of 14 days the conversion was in the range of 0.03to 0.06 depending on the way it was calculated. In other words, this isa reversible process, with the reaction heavily displaced in thesynthesis direction.

The experiments indicate that very high conversion and very high opticalpurity are achieved by using an enzyme immobilised in a gel inaccordance with this invention. The gel may be re-used several times,with long intermediate storage. Compared with previous enzymeimmobilising methods, this invention has the advantage of not requiringthe use of any foreign substances, such as surfactants. This enablesnon-toxic and environment-friendly enzyme-catalysing processes to beimplemented. At the same time, the risks of competing enzyme reactionsare reduced. In addition, the process is energy-saving.

Compared with prior art, the gel in accordance with this invention has anumber of appreciable advantages in being mechanically divisible intofragments, which dimensionally are at least almost stable during theactual division and subsequent use (for instance in stereoselectivesynthesis). The possibility of selecting the size and the shape of thegel fragment to be added to the reaction mixture provides a practicalsolution. After the reaction has been performed, recovery of the gelwill be perfectly simple if the gel has the form of one single piece ora few pieces. A gel in the form of small (approx. 1 mm) beads requires aspecific filtrating step, necessarily involving a given particle loss,since these beads are likely to constitute a polydisperse mixture ofbeads of varied sizes. In the recovery, the smallest fraction mayperhaps pass through the filter and be lost. A gel which for instancecan be cut into thin slices combines two advantageous properties: alarge active surface (as opposed to cast cubes) with simultaneous easeof recovery (as opposed to polydisperse beads). Moreover, the cuttingprovides a section surface with open pores, thus ensuring contactbetween the enzyme and the reaction solution. In contrast, the castingmethod yields gels with closed pores. Also the preparation of droplets(beads) is likely to produce a surface having closed pores, because thehydrophobic parts of the gelatiniser will be directed outwards towardsthe surface of the droplet.

Although the invention has been described here with reference to aparticular type of reaction and a specific gelatiniser, the invention isby no means limited to these conditions. On the contrary, the inventionshall be considered to comprise all the applications and embodimentsdisclosed in the accompanying claims.

What is claimed is:
 1. A gel containing an immobilized enzyme,comprising a water-soluble enzyme, a water-soluble gelatin and water,wherein a ratio of the amount of gelatin to the amount of water isselected such that the gel is capable of being mechanically divisibleinto dimensionally substantially stable fragments at a temperature whichmay reach a lower limit of the gelation temperature range of thegelatin, wherein said gel has a 30 to 50% gelatin concentration.
 2. Thegel of claim 1, wherein said gel has a 30 to 44% gelatin concentration.3. The gel of claim 1, wherein said enzyme is lipase.
 4. The gel ofclaim 1, wherein said lower limit of the gelation temperature range ofthe gelatin is 30° C.
 5. A process for preparing the gel of claim 1,comprising i) adding water to a water-soluble enzyme and a water-solublegelatin to form a mixture; ii) heating said mixture to a temperature atwhich the gelatin dissolves, thereby forming a solution; and iii)cooling said solution until it forms a gel capable of being mechanicallydivided into dimensionally substantially stable fragments at atemperature that may reach a lower limit of the gelation temperaturerange of the gelatin, wherein said gel has a 30 to 50% gelatinconcentration.
 6. The process of claim 5, wherein said lower limit ofthe gelation temperature range of the gelatin is 30° C.
 7. A process forperforming an enzyme-catalyzed reaction, comprising adding a fragment ofthe gel of claim 1 to a reaction mixture consisting of reactantsdissolved in a hydrophobic solvent; so as to catalyze a reaction andthereby produce a desired reaction product.
 8. The process of claim 7,wherein said enzyme comprises lipase.
 9. The process of claim 8, whereinsaid enzyme-catalyzed reaction comprises a synthesis of optically activeisomers of an ester from an alcohol and an acid, with either the alcoholor the acid being a racemic mixture or an enantiomer.
 10. The process ofclaim 8, wherein said enzyme-catalyzed reaction is a hydrolysis of anester which is either a racemate or a pure enantiomer.
 11. The processof claim 8, wherein said enzyme-catalyzed reaction is atransesterfication.